CN104204801B - Immunological analysis method and reagent - Google Patents

Abstract

Disclosed are: an immunological analysis method which can measure an antigen with high sensitivity and high accuracy; and a reagent for use in the method. In the immunological analysis method, an antigen-antibody reaction and/or a measurement is carried out in the presence of a polycarboxylic acid-type surfactant. The immunological analysis reagent for use in the method is characterized by comprising a polycarboxylic acid-type surfactant. By employing such a simple means that a polycarboxylic acid-type surfactant is allowed to exist in a reaction and/or measurement system, it becomes possible to inhibit a non-specific reaction effectively in a high-sensitivity immunological analysis, whereby an antigen can be measured accurately and specificity can be improved in an immunological analysis.

Description

Immunoassay methods and reagents

technical field

The present invention relates to immunoassay methods and reagents for use in said methods.

Background technique

Immunoassay methods are widely used in clinical examinations of serum, plasma, urine, feces, cerebrospinal fluid, etc. In recent years, automatic analyzers that automatically perform reactions to measurements all at once have been widely used because they can be measured simply and quickly.

Immunoassay methods are known as measurement methods that utilize antigen-antibody reactions and have high specificity. However, there is a problem of non-specific reactions such as false positives and false negatives depending on the sample. There is a problem of showing a measured value different from the true value, for example, there is a case where a factor that recognizes an antibody and reacts exists in a sample, and in this case a positive measured value is obtained even if the antigen to be measured is not present in the sample, and another On the other hand, there are cases where a factor that suppresses the antigen-antibody reaction exists in the sample. In this case, a negative measurement value is obtained even if the antigen to be measured is present in the sample.

As means for suppressing nonspecific reactions, polymers in which human IgM natural antibodies, aromatic monomers having a sulfone group or salts thereof are added and polymerized are known (see Patent Documents 1 and 2). However, these additives are sometimes insufficient, and it is particularly difficult to suppress non-specific reactions in low-concentration regions. Further, with highly sensitive reagents, nonspecific reactions tend to occur due to increased antibody reactivity.

prior art literature

patent documents

Patent Document 1: Patent No. 4065600

Patent Document 2: Patent No. 4580180.

Contents of the invention

The problem to be solved by the present invention

An object of the present invention is to provide an immunoassay method capable of highly sensitive and accurate antigen measurement in an immunoassay and a reagent used in the method.

means of solving problems

As a result of earnest studies by the inventors of the present application, it has been found that non-specific reactions can be easily suppressed even in high-sensitivity measurements by including a polycarboxylic acid-type surfactant in an immunoassay.

That is, the present invention relates to the following 1)-9).

1) Immunoassay method, which performs antigen-antibody reaction and/or measurement in the presence of a polycarboxylic acid-type surfactant.

2) The method of 1), wherein the polycarboxylic acid-type surfactant is: (1) a copolymer of maleic acid and/or maleic anhydride or a salt thereof and (2) diisobutylene.

3) The method of 1) or 2), wherein, in the reaction system and/or measurement system where the polycarboxylic acid surfactant exists, the concentration of the polycarboxylic acid surfactant is 0.001%-3%.

4) The method according to any one of 1) to 3), wherein the measurement is performed from the start of the antigen-antibody reaction to the end of the measurement in the presence of the polycarboxylic acid-type surfactant.

5) The method according to any one of 1)-4), wherein the immunoassay method is an immunoagglutination method.

6) The method of 5), wherein the immunoagglutination method is a latex agglutination method.

7) The immunoassay reagent used in the method of 1), comprising a polycarboxylic acid-type surfactant.

8) The reagent of 7), wherein the polycarboxylic acid-type surfactant is: (1) a copolymer of maleic acid and/or maleic anhydride and (2) diisobutylene.

9) The reagent of 7) or 8), which is a reagent for immunoagglutination, and further contains a reagent for immunoagglutination.

10) The reagent of 9), which is a reagent for latex agglutination and further contains a reagent for latex agglutination.

The effect of the invention

According to the method of the present invention, non-specific reactions can be effectively suppressed even in highly sensitive immunoassays by the simple means of allowing the polycarboxylic acid type surfactant to exist in the reaction and/or assay system. In immunoassays, it is possible to Correctly determine the antigen and improve specificity.

Description of drawings

FIG. 1 shows calibration curves prepared in Examples and Comparative Examples described below.

Fig. 2 shows the correlation of the immunoassay method of the following examples with the measurement results of the immunoassay method using a commercially available reagent (existing reagent) known to suppress non-specific reactions.

FIG. 3 is an enlarged display of the low-concentration region in FIG. 2 .

Fig. 4 shows the correlation between the measurement results of the immunoassay method of the following comparative example and the immunoassay method using conventional reagents.

FIG. 5 is an enlarged display of the low-concentration region in FIG. 4 .

Fig. 6 shows the correlation between the measurement results of the immunoassay method of the following comparative example and the immunoassay method using conventional reagents.

FIG. 7 is an enlarged display of the low-concentration region in FIG. 6 .

Fig. 8 shows the correlation between the measurement results of the immunoassay method of the following comparative example and the immunoassay method using conventional reagents.

FIG. 9 is an enlarged display of the low-concentration region in FIG. 8 .

Fig. 10 shows the correlation between the measurement results of the immunoassay method of the following examples and the immunoassay method using conventional reagents.

FIG. 11 is an enlarged display of the low-concentration region in FIG. 10 .

Fig. 12 shows the correlation between the measurement results of the immunoassay method of the following examples and the immunoassay method using conventional reagents.

FIG. 13 shows an enlarged display of the low-concentration region in FIG. 12 .

Fig. 14 shows the correlation between the measurement results of the immunoassay method of the following examples and the immunoassay method using conventional reagents.

FIG. 15 shows an enlarged display of the low-concentration region in FIG. 14 .

Fig. 16 shows the correlation between the measurement results of the immunoassay method of the following example and the immunoassay method using conventional reagents.

FIG. 17 shows an enlarged display of the low concentration region in FIG. 16 .

Fig. 18 shows the correlation between the measurement results of the immunoassay method of the following comparative example and the immunoassay method using conventional reagents.

FIG. 19 is an enlarged display of the low-concentration region in FIG. 18 .

Fig. 20 shows the correlation between the measurement results of the immunoassay method of the following example and the immunoassay method using conventional reagents.

FIG. 21 shows an enlarged display of the low-concentration region in FIG. 20 .

Fig. 22 shows the correlation between the measurement results of the immunoassay method of the following example and the immunoassay method using conventional reagents.

FIG. 23 shows an enlarged display of the low-concentration region in FIG. 22 .

Fig. 24 shows the correlation between the measurement results of the immunoassay method of the following example and the immunoassay method using conventional reagents.

FIG. 25 is an enlarged display of the low-concentration region in FIG. 24 .

detailed description

The method of the present invention is explained below. In addition, "%" in this specification means a mass basis (w/v%) unless otherwise specified.

The immunoassay method of the present invention uses an immunoassay reagent that reacts immunologically to a substance to be measured in a sample, conducts an antigen-antibody reaction, and measures the obtained reactant, which is characterized in that the reaction is carried out in the presence of a polycarboxylic acid-type surfactant. response and/or assay.

The means of immunoassays are known per se. The immunoassay method applicable to the method of the present invention can be any known immunoassay method, among which the immunoagglutination method is preferred, and the latex agglutination method using latex particles as insoluble carrier particles is especially preferred. Methods for detecting agglutination of sensitized particles in immunoagglutination are known, and known methods, such as methods for detecting absorbance or light scattering caused by agglutination of sensitized particles, can also be used in the present invention. Examples include immunoturbidimetry (TIA method, latex agglutination method), colorimetry, RPLA method, CL method, and immunochromatography. Nephelometric methods and colorimetric methods with high sensitivity and high quantitative accuracy are preferred. .

The insoluble carrier particles to be used in the form of this immunoassay method are not particularly limited, and may be known particles conventionally used in immunoassay reagents. For example, particles such as latex particles such as polyethylene or polystyrene, alumina particles, silica particles, colloidal gold, and magnetic particles may be mentioned. Latex particles, especially polystyrene latex particles, are preferably used among these insoluble carriers. Immunoagglutination is known as a method for optically detecting agglutination of sensitized particles induced by antigens or antibodies, and turbidimetric or colorimetric methods are preferably used for detection. For example, light from visible light to near-infrared region is irradiated from the outside of the cuvette, such as generally 300-1000nm, preferably 500-900nm light, and the change in absorbance or the intensity of scattered light is detected to determine the degree of aggregation of the sensitized particles. The latex particles are particularly preferably polystyrene latex particles. The size of the latex particles is not particularly limited, but the particle diameter is preferably 30-600 nm.

An antibody or an antigen-binding fragment thereof that immunoreacts with an antigen to be measured is immobilized on the above-mentioned latex particles. The method of immobilization is also known, and is carried out by known methods such as physical adsorption and covalent bonding. When the obtained sensitized particle suspension is mixed with a test sample, the sensitized particles aggregate due to the substance to be measured (antigen) contained in the test sample, and the absorbance of the sensitized particle suspension changes. The amount of change (endpoint method) or rate of change (ratio method) of this absorbance is measured. A plurality of standard samples containing the antigen to be measured at various known concentrations are prepared, and the amount of change or the rate of change in absorbance is measured for them by the method described above. Draw the calibration curve with the concentration of the antigen to be measured in the standard sample as the horizontal axis and the measured absorbance change amount or rate of change as the vertical axis. For unknown samples to be tested, the change amount or rate of change of absorbance is measured by the same method, and the measurement result is compared with the above calibration curve, so that the antigen in the sample to be tested can be quantified.

In addition, various automatic devices for performing the immunoagglutination method are commercially available, and it can be easily and simply carried out by using a commercially available automatic device for the immunoagglutination method.

The substance to be measured in the immunoassay in the present invention is not limited in any way as long as it can be measured by immunoassay. When the substance to be measured is an antigen, examples include: CRP (C-reactive protein), prostate-specific antigen, iron protein, β-2 microglobulin, myoglobin, hemoglobin, albumin, creatinine and other protein markers, IgG, IgA, IgM and other immunoglobulins, various tumor markers, LDL, HDL, TG and other lipoproteins, Influenza A virus, influenza B virus, RS virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus ), astrovirus, HAV, HBs, HCV, HIV, EBV and other viral antigens, chlamydia trachomatis, hemolytic streptococcus, bordetella pertussis, helicobacter pylori pylori), leptospira, treponema palidum, toxoplasma gondii, borrelia, legionella, anthrax bacillus, MRSA and other bacteria Antigens, toxins produced by bacteria, mycoplasma lipid antigens, peptide hormones such as human chorionic gonadotropin, steroid hormones such as steroids, physiologically active amines such as epinephrine and morphine, vitamins such as vitamin B, prostaglandins class, tetracycline and other antibiotics, pesticides, environmental hormones, etc., but not limited thereto. Preferable examples include antigens such as CRP, prostate-specific antigen, ferritin, β-2 microglobulin, and hemoglobin.

When the substance to be measured is an antibody, examples include: toxins, peptide hormones, steroids, physiologically active amines, vitamins, Antibodies that react specifically to antigens such as antibiotics, pesticides, and environmental hormones.

The sample used in the immunoassay is not particularly limited, as long as it contains the substance to be measured, examples include: blood, serum, plasma, urine, feces, saliva, interstitial fluid, cerebrospinal fluid, swab fluid and other body fluids, etc. or their dilutions, preferably blood , serum, plasma, urine, feces, cerebrospinal fluid or their dilutions.

As described above, the method of the present invention is characterized in that the antigen-antibody reaction and/or measurement are performed in the presence of a polycarboxylic acid-type surfactant in the reaction system and/or measurement system. A "polycarboxylic acid-type surfactant" is a surfactant having a plurality of carboxyl groups in one molecule or a salt thereof, and/or an acid anhydride group (at least a part of the acid anhydride group is hydrolyzed in water to generate a carboxyl group, and therefore has a plurality of carboxyl groups in one molecule in water) ) is a polymeric surfactant known as one of the anionic surfactants, and various polycarboxylic acid-type surfactants are known and commercially available and used industrially. The present invention can be applied to any type of polycarboxylic acid, among which: (1) copolymer of maleic acid and/or maleic anhydride and (2) diisobutylene, and/or salts thereof are preferred. Here, the salt is not particularly limited, and sodium salt is preferred. Since the copolymer or its salt is widely used in industry and is also commercially available, it is also possible to preferably utilize a commercially available product in the present invention (see Examples below).

The weight average molecular weight of the polycarboxylic acid type surfactant is not particularly limited, but is preferably about 10 million to 50 thousand.

In the method of the present invention, for the polycarboxylic acid type surfactant, at any stage from the beginning of the antigen-antibody reaction to the detection and quantification of the amount of antigen-antibody reaction, the polycarboxylic acid type surfactant is in the reaction and/or measurement system (also (referred to as "reaction and measurement system") may be included, and it is preferably included in the period from the initiation of the antigen-antibody reaction to the detection and quantification. Therefore, the polycarboxylic acid surfactant is preferably added to the reaction system before the antigen-antibody reaction or at the same time as the antigen-antibody reaction. Specifically, it can be added when diluting the sample, or when the antibody or antigen is mixed with the sample.

In addition, polycarboxylic acid surfactants may be contained in various reagents used in immunoassays, and the present invention also provides immunoassay reagents containing the polycarboxylic acid surfactants. Here, various reagents used in immunoassays include, for example, sample diluent, antibody/antigen diluent, immobilized antibody/antigen, sensitized particle suspension, washing solution, enzyme solution, matrix solution, Examples of immunoassay reagents containing polycarboxylic acid-type surfactants, such as standard solutions of test substances for calibration curves, include: reagents in which polycarboxylic acid-type surfactants are added to these reagents, for example, buffer solutions for diluting samples , reagents containing antibodies or antigens, etc., which contain polycarboxylic acid-type surfactants.

Also, for example, a polycarboxylic acid-type surfactant can be included in an immunoagglutination reagent containing latex particles (sensitized particles) in which antibodies or antigens are immobilized (sensitized). At this time, the concentration of sensitized particles in the immunoassay reagent is not particularly limited, and is preferably 0.01-0.5%. The amount of antibody and antigen in the sensitized particle suspension can be determined according to conventional methods, and is not particularly limited. For example, in the case of antibody-sensitized latex, the preferred amount of antibody in the latex suspension is 0.01-2.0 mg/mL.

From the aspect of non-specific reaction inhibition, the concentration of the polycarboxylic acid surfactant in the reaction and measurement system is preferably 0.001-3%, more preferably 0.005-1%. Therefore, when the polycarboxylic acid-type surfactant is previously contained in the immunoassay reagent, it may be included in the immunoassay reagent so that the concentration in the reaction and/or measurement system becomes the above-mentioned concentration.

The blank sample used in the immunoassay is not particularly limited as long as it does not contain the substance to be measured, and is preferably purified water, physiological saline, buffer, negative sample or their dilutions.

Non-specific reactions are suppressed when polycarboxylic acid-type surfactants are present in the reaction and/or assay system as disclosed in the Examples below. Also, the specificity is predominantly improved compared to the absence of the polycarboxylic acid-type surfactant. Therefore, by applying the method of the present invention, the performance of the reagent can be improved compared to conventional ones, especially in highly sensitive reagents that are prone to non-specific reactions.

Hereinafter, based on an Example and a comparative example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Embodiment 1, comparative example 1-3

(1) Preparation of reagents

Using an antibody against ferritin, a measurement reagent by the immunoagglutination method was prepared as follows.

i) Sensitized particles loaded with 0.03 mg of anti-ferritin antibody with respect to 1 mL of polystyrene latex suspension with an average particle diameter of 300 nm were suspended in a buffer solution (tris, pH 8.0) so as to become 0.04% , and the preparation of latex suspension.

ii) A polycarboxylic acid-type surfactant (maleic acid-diisobutylene copolymer sodium) was added to a buffer solution (tris, pH 8.5) to prepare the following reagent A (Example 1). As Comparative Examples 1-3, reagents B-D were prepared without adding any substance or further adding other additives.

【Table 1】

example Reagent Additive (concentration) Example 1 A ＋Maleic acid・Sodium diisobutylene copolymer*1 1.0% Comparative example 1 B none Comparative example 2 C +Normal rabbit globulin 3g/L Comparative example 3 D. ＋Sodium polystyrene sulfonate*2 1.0%

*1: Polycarboxylic acid-type surfactant (anionic surfactant) "polystar OM" (trade name, commercially available from NOF Corporation)

*2: Anionic surfactant "PS-5" (trade name, commercially available from TOSOH CORPORATION).

(2) Measurement using an automatic analyzer

As an automatic analyzer, Hitachi Model 7180 automatic analyzer was used for automatic measurement by the endpoint method.

Using reagents A-D described above, 24 serum samples containing RF positive samples known to cause non-specific reactions were assayed. 100 μL of the above-prepared buffer solutions of reagents A to D were added to 10.0 μL of the sample solution, and the mixture was stirred and mixed at 37°C. After standing for 5 minutes, 100 µL of the latex suspension was added, and further stirred and mixed at 37°C. The agglutination reaction for about 5 minutes was measured by the change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.

(3) Compared with the sensitivity of existing reagents

The measurement sensitivity of the above-mentioned Example 1 and Comparative Examples 1-3 is compared with that of the existing commercially available latex reagent FER-Latex X2 "Denka Seiken" (hereinafter referred to as "existing reagent") known to suppress non-specific reactions. sensitivity for comparison. The results are shown in Figure 1.

As shown in Figure 1, in the methods of Example 1 and Comparative Examples 1-3, compared with the method using existing reagents, if the concentration of ferritin is the same, the amount of change in absorbance becomes larger, and the amount of change in the absorbance of Example 1 and Comparative Example 1 The method of -3 has high sensitivity.

(4) Comparison of specificity with existing reagents

The measurement results of the above-mentioned Example 1 and Comparative Examples 1-3 were compared with the measurement results of the method using the above-mentioned conventional reagents. The results are shown in Figures 2-9, respectively.

Existing reagents are known as reagents that suppress non-specific reactions. Comparing Fig. 2 and Fig. 3 showing the results of Example 1 and Fig. 4 to Fig. 9 showing the results of each comparative example shows that in the highly sensitive immunoassay method, by adding a polycarboxylic acid-type surfactant, Non-specific reactions are suppressed and correlation with assay results when using existing reagents is increased. showed an increase especially in low concentration regions.

Embodiment 2-5, comparative example 4

(1) Preparation of reagents

Using an antibody against ferritin, a measurement reagent by the immunoagglutination method was prepared as follows.

i) Sensitized particles loaded with 0.03 mg of anti-ferritin antibody with respect to 1 mL of polystyrene latex suspension with an average particle diameter of 300 nm were suspended in a buffer solution (tris, pH 8.0) so as to become 0.04% , to prepare a latex suspension.

ii) A polycarboxylic acid-type surfactant (sodium maleate-diisobutylene copolymer, "polystar OM" (trade name, commercially available from NOF Corporation)) was added to a buffer (tris, pH 8.5) to prepare Reagents E-H (Examples 2-5) below. As Comparative Example 4, Reagent I without adding any substance was prepared.

【Table 2】

example Reagent Additive (concentration) Example 2 E. ＋Sodium maleic acid and diisobutylene copolymer 0.1% Example 3 f ＋Sodium maleic acid and diisobutylene copolymer 0.5% Example 4 G ＋Sodium maleic acid and diisobutylene copolymer 1.0% Example 5 h ＋Sodium maleic acid and diisobutylene copolymer 2.0% Comparative example 4 I none

(2) Measurement using an automatic analyzer

As an automatic analyzer, Hitachi Model 7180 automatic analyzer was used for automatic measurement by the endpoint method.

Using the aforementioned reagents E-I, 24 serum samples containing RF positive samples known to cause non-specific reactions were assayed. 100 μL of the above-prepared buffer solution of reagent E-I was added to 10.0 μL of the sample solution, and the mixture was stirred and mixed at 37° C. After standing for 5 minutes, 100 µL of the latex suspension was added, and further stirred and mixed at 37°C. The agglutination reaction for about 5 minutes was measured by the change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.

(3) Comparison with existing reagents

The measurement results of the above-mentioned Examples 2-5 and Comparative Example 4 were compared with the measurement results of the method using the above-mentioned conventional reagents. The results are shown in Figures 10-19, respectively.

Existing reagents are known as reagents that suppress non-specific reactions. Comparing Figures 10-17 showing the results of Examples 2-5, and Figures 18-19 showing the results of Comparative Example 4, shows that in the highly sensitive immunoassay method, by adding polycarboxylic acid-type surfactant Reagents, non-specific reactions are suppressed, and the correlation with measurement results when using existing reagents is increased. showed an increase especially in low concentration regions.

Embodiment 6, 7, comparative example 5

(1) Preparation of reagents

Using an antibody against ferritin, a measurement reagent by the immunoagglutination method was prepared as follows.

i) Sensitized particles loaded with 0.03 mg of anti-ferritin antibody with respect to 1 mL of polystyrene latex suspension with an average particle diameter of 300 nm were suspended in a buffer solution (tris, pH 8.0) so as to become 0.04% , to prepare a latex suspension.

ii) A polycarboxylic acid-type surfactant was added to a buffer solution (tris, pH 8.5) to prepare the following reagent J (Example 6) and reagent K (Example 7). As Comparative Example 5, reagent L to which nothing was added was prepared.

【table 3】

example Reagent Additive (concentration) Example 6 J ＋Maleic acid・Sodium diisobutylene copolymer*1 1.0% Example 7 K ＋Polycarboxylic acid type surfactant*2 1.0% Comparative Example 5 L none

*1: Polycarboxylic acid-type surfactant (anionic surfactant) "polystar OM" (trade name, commercially available from NOF Corporation)

*2: Polycarboxylic acid-type surfactant (trade name "Demol EP" (DEMOL EP), commercially available from Kao Corporation).

(2) Measurement using an automatic analyzer

As an automatic analyzer, Hitachi Model 7180 automatic analyzer was used for automatic measurement by the endpoint method.

Twenty-four serum samples containing RF-positive samples known to cause non-specific reactions were assayed using the aforementioned reagents J-L. 100 µL of the above-prepared buffer solution of Reagent J-L was added to 10.0 µL of the sample solution, and the mixture was stirred and mixed at 37°C. After standing for 5 minutes, 100 µL of the latex suspension was added, and further stirred and mixed at 37°C. The agglutination reaction for about 5 minutes was measured by the change in absorbance, and the ferritin concentration of each sample was calculated from the calibration curve.

(3) Comparison with existing reagents

The measurement results of the above Examples 6 and 7 and Comparative Example 5 were compared with the measurement results of the method using the above conventional reagents. The results are shown in Figures 20-25, respectively.

Existing reagents are known as reagents that suppress non-specific reactions. Comparing Figure 20-Figure 23 showing the results of Examples 6-7, and Figure 24-Figure 25 showing the results of Comparative Example 5, shows that in the highly sensitive immunoassay method, by adding polycarboxylic acid-type surfactant Reagents, non-specific reactions are suppressed, and the correlation with measurement results when using existing reagents is increased. showed an increase especially in low concentration regions.

Claims (5)

1. immune analysis method, it carries out antigen-antibody reaction and/or mensure in the presence of polycarboxylate-type surfactant, its In, described polycarboxylate-type surfactant is：(1) EVA of maleic acid and/or maleic anhydride and (2) diisobutylene, and/or Its salt,

Wherein, described immune analysis method is immune agglutination method.

2. the method described in claim 1, wherein, in reaction system and/or the survey of the presence of described polycarboxylate-type surfactant Determine in system, the concentration of described polycarboxylate-type surfactant is 0.001%-3%.

3. the method any one of claim 1-2, wherein, in the presence of described polycarboxylate-type surfactant, from Antigen-antibody reaction proceeds by and terminates to mensure.

4. the method any one of claim 1-2, wherein, described immune agglutination method is latex agglutination.

5. the method described in claim 3, wherein, described immune agglutination method is latex agglutination.